JP6838612B2 - Inspection chip and inspection system - Google Patents

Description

The present invention relates to an inspection chip having a structure on the side surface. The present invention also relates to an inspection system using an inspection chip having a structure on a side surface.

Biochemical reactions such as antigen-antibody reaction are used in biochemical tests. For example, in fluoroimmunoassay (FIA), a labeling substance containing a fluorescent substance is bound to a substance to be detected (antigen) to fluorescently label the substance to be detected. Then, the fluorescently labeled substance to be detected is irradiated with excitation light, the fluorescence emitted from the fluorescent substance is detected, and the amount of the substance to be detected is specified from the intensity of the fluorescence. Among such FIA, surface plasmon-field enhanced fluorescence spectroscopy (SPFS) is known as a method capable of detecting a substance to be detected with particularly high sensitivity.

In SPFS, a first trap (for example, a primary antibody) that specifically binds to a substance to be detected is immobilized on a metal membrane to form a reaction field for capturing the substance to be detected. For example, Patent Document 1 discloses an SPFS apparatus including a well-type inspection chip (sensor structure 22) in which a reaction field is arranged on the bottom surface of a well (a bottomed recess for containing a liquid). .. In the inspection chip, the well is formed by fixing a well member having a through hole on a metal film formed on a light-transmitting dielectric member, and the reaction field is formed on the bottom surface of the well. It is arranged on the metal film constituting the above. Then, by introducing a sample (sample or the like) that can contain the substance to be detected into this well, the substance to be detected is bound to the first trap that is immobilized on the metal film and forms the reaction field. .. Then, by introducing a fluorescently labeled second trap (for example, a secondary antibody) into the well, the second trap is further bound to the substance to be detected bound to the first trap. That is, the substance to be detected is indirectly fluorescently labeled. When the metal film is irradiated with excitation light from the dielectric member side in this state, the fluorescent substance is excited by an electric field enhanced by surface plasmon resonance (SPR) and emits fluorescence. In the SPFS apparatus disclosed in Patent Document 1, the fluorescence emitted from the fluorescent substance passes through the liquid surface of the liquid in the well and is detected by a detection unit arranged above the well.

International Publication No. 2012/157403

As described above, in the inspection chip disclosed in Patent Document 1, since a metal film is formed on the bottom surface of the well and the reaction field is arranged, the tip of the liquid feeding device is used when removing a liquid such as a reagent in the well. May come into contact with the metal film or reaction field and damage them. Therefore, the tip of the liquid feeding device cannot be pressed against the bottom surface of the well, and it is difficult to sufficiently remove the liquid in the well. If the liquid such as the reagent is not sufficiently removed and remains in the well, various reactions do not proceed properly and the detection accuracy deteriorates.

Further, as described above, in the SPFS apparatus disclosed in Patent Document 1, the detection unit is arranged above the well and detects the fluorescence that has passed through the liquid surface of the liquid in the well. The detection result may be affected by the meniscus or the bubbles existing on the liquid surface. If the fluorescence detection result is affected by meniscus, bubbles, or the like, the detection accuracy will decrease.

That is, there is a problem that the detection accuracy is lowered in the inspection chip in which the reaction field is provided on the bottom surface of the well as in the prior art.

On the other hand, in order to sufficiently supply a liquid such as a reagent to the reaction field and carry out the reaction efficiently, it is necessary to stir the liquid contained in the test chip. As a general stirring method, stirring by circular motion (hereinafter referred to as circular motion stirring) is known. FIG. 1 is a schematic diagram for explaining the circular motion of the conventional inspection tip 60x installed on the rotating body 99 of the stirring device. The upper part of FIG. 1 shows a side view of the inspection tip 60x installed on the rotating body 99 of the stirring device when performing circular motion stirring. The lower part of FIG. 1 shows a schematic view of the inspection tip 60x as viewed from the opening side when performing circular motion stirring. In the lower part of FIG. 1, for convenience of notation, the inspection chip 60x is represented by a straight line, and the motion locus of the tip 66b of the inspection chip 60x that comes into contact with the rotating body 99 and receives the circular motion of the rotating body 99 is broken. It is written in.

In circular motion stirring, one end of the inspection chip 60x on the opening side is fixed by a fixing member (not shown), and the other end of the inspection chip 60x on the bottom surface side is installed on the rotating body 99 of the stirring device as shown in FIG. Then, the inspection chip 60x is made to make a circular motion with the rotating body 99 which makes a circular motion. In the conventional well-type inspection chip 60x (for example, a test tube), the inspection chip 60x has a symmetrical structure, and rotates in contact with the center of gravity G1 in a state where a liquid such as a reagent is contained and the rotating body 99. Since the tip 66b that receives the circular motion of the body 99 exists on the same axis in the length direction (vertical direction in FIG. 1) of the inspection chip 60x, both have the same motion trajectory as shown in the lower part of FIG. As shown, the inspection chip 60x can perform a stable circular motion along with the circular motion of the rotating body 99.

However, in order to solve the above-mentioned problem that the detection accuracy is lowered when the test chip provided on the bottom surface of the well is used, if the reaction field is provided at a position other than the bottom surface of the well, Along with this, the dielectric member and the like have to be moved, and the structure of the inspection chip becomes asymmetric. For example, the case where a test chip having a reaction field on the side surface is used will be described below.

FIG. 2 is a schematic view for explaining the circular motion of the inspection tip 60y installed on the rotating body 99 of the stirring device and having the reaction field on the side surface. The upper part of FIG. 2 shows a side view of the inspection tip 60y installed on the rotating body 99 of the stirring device when performing circular motion stirring. The lower part of FIG. 2 shows a schematic view when the inspection tip 60y is viewed from the opening side when performing circular motion stirring. In the lower part of FIG. 2, for convenience of notation, the inspection chip 60y is represented by a straight line, and the tip 66b of the inspection chip 60y that comes into contact with the rotating body 99 and receives the circular motion of the rotating body 99, the reagent, and the like. The motion loci of the center of gravity G2 of the inspection chip 60y in the state where the liquid is contained are shown by broken lines.

As shown in FIG. 2, in the inspection chip 60y having a reaction field on the side surface, the inspection chip 60y has an asymmetric structure and is in contact with the center of gravity G2 in a state where a liquid such as a reagent is contained and the rotating body 99. Since the tip 66b of the inspection chip 60y that receives the circular motion of the rotating body 99 is separated from each other and exists on different axes in the length direction (vertical direction in FIG. 2) of the inspection chip 60y, the lower part of FIG. As shown in the above, the two show different motion trajectories, and the circular motion of the inspection chip 60y is affected by this and becomes unstable. Specifically, the inspection tip 60y may fall from the stirrer. Further, even when the inspection chip 60y does not fall from the stirring device, the liquid such as the reagent in the inspection chip 60y cannot be efficiently stirred, and even if stirring is performed, the reagent or the like in the inspection chip 60y is not stirred. It may not be expected that the liquid of the above will sufficiently supply the liquid such as a reagent to the reaction field without forming a so-called vortex.

That is, in the prior art, it has not been possible to further improve the detection accuracy of the substance to be detected and to achieve stable and efficient stirring at the same time.

The inspection chip of the present invention is an inspection chip for accommodating a liquid inside and stirring the liquid by a circular motion at the bottom end, and is provided on a well body for accommodating the liquid and a side surface of the well body. It has a side wall member that is arranged, and the bottom end of the well body comes into contact with a rotating member for circularly moving the bottom end at a position biased from the center line of the well body to the side wall member. Has a bottom structure.

The inspection system of the present invention is an inspection system using the inspection chip of the present invention, and is a light source that irradiates the inspection chip with light and a detection unit for measuring the light to be measured emitted from the inspection chip. And a stirring device having the rotating member.

According to the present invention, it is possible to further improve the detection accuracy of the substance to be detected and simultaneously realize stable and efficient stirring.

It is a schematic diagram for demonstrating the circular motion of the conventional inspection tip installed in the rotating body of a stirrer. It is a schematic diagram for demonstrating the circular motion of the conventional inspection tip which is installed in the rotating body of a stirrer and has a structure on the side surface. It is a schematic diagram which shows the structure of a biochemical inspection system. FIG. 4A is a perspective view of the inspection chip. FIG. 4B is a perspective view of the well body. FIG. 4C is a perspective perspective view of the well body. It is a schematic diagram which shows the structure of the side wall member. It is a schematic diagram when the inspection chip is seen from the 1st opening side. It is a block diagram of a control calculation part. It is a schematic diagram for demonstrating the circular motion of the inspection tip installed in the rotating body of a stirrer. It is a flowchart for demonstrating operation of a biochemical inspection system.

Hereinafter, one embodiment for carrying out the present invention will be described with reference to the drawings. The present embodiment is a biochemical inspection system, in which a plurality of measuring units that perform individual steps constituting one inspection are arranged in order on the production line, and the inspection chips proceed along the production line. As the individual steps are performed in order and the inspection progresses, a continuous form is adopted in which a plurality of inspection chips can be introduced one after another so that a plurality of inspections can be performed almost at the same time. However, the present invention is not limited to the continuous form. For example, it is possible to adopt a discontinuous form in which the individual steps constituting one inspection are performed at the same position and the progress of the inspection does not depend on the progress of the inspection chip.

Although the present embodiment employs a method using SPFS as a biochemical test, the present invention is not limited to the method using SPFS. For example, methods such as surface plasmon resonance (SPR) and general fluorescence immunoassay can also be adopted. However, the type and mode of inspection are not limited, and if the use of an asymmetric inspection chip is required, the detection accuracy of the substance to be detected is further improved, and stable and efficient stirring is performed. And can be obtained at the same time.

(Biochemical testing system)
FIG. 3 is a schematic view showing the configuration of the biochemical inspection system A according to the present embodiment. The biochemical test system A is a system for performing a biochemical test using SPFS. Specifically, the biochemical inspection system A captures the substance to be detected by the first trapping object immobilized on the metal film, and fluoresces the substance to be detected captured by the first trapping substance by the fluorescent substance. The labeled second trap is attached to fluorescently label the substance to be detected. After that, the metal film is irradiated with excitation light to generate an enhanced electric field based on surface plasmon resonance in the vicinity of the metal film, and the fluorescence emitted from the fluorescent substance excited by the enhanced electric field is detected to detect the presence or amount of the substance to be detected. To measure.

As shown in FIG. 3, the biochemical inspection system A includes a vibration excitation unit 10, a light projecting unit 20, a liquid feeding / transporting unit 30, a detecting unit 40, and a control calculation unit 50. 20 is configured to irradiate the inspection chip arranged in the excitation unit 10 with excitation light, and the detection unit 40 is configured to detect the fluorescence emitted from the inspection chip. The specific configuration of each part will be described below.

(Vibration section)
The vibrating unit 10 includes a stirring device (not shown) that stirs the liquid contained in each of the inspection chips 60a, 60c and 60d by rotational vibration at the positions 10a, 10c and 10d corresponding to the inspection chips 60a, 60c and 60d, respectively. Be prepared. The stirring device is arranged at a position that does not obstruct the optical path such as excitation light, fluorescence, and plasmon scattered light, and has an eccentric rotating body. The rotating body performs rotational vibration in contact with the inspection chip to apply rotational vibration in the circumferential direction to the inspection chip and agitate the liquid contained in the inspection chip. However, the stirring device is not limited to one having an eccentric rotating body, and any stirring device may be used as long as it can stir the liquid contained in the inspection chip by applying rotational vibration to the inspection chip.

By agitating the liquid contained in the inspection chip by the agitator, the reaction and cleaning of each step in the biochemical inspection can be efficiently performed. From the viewpoint of efficiently stirring the liquid in the inspection chip, the stirring device preferably applies rotational vibration to the inspection chip at the natural frequency of the inspection chip containing the liquid or a vibration frequency before and after the natural frequency. Further, rotational vibration may be applied to the inspection chip while sequentially switching between different natural frequencies (n-th order natural frequency and m-th order natural frequency, where n and m are positive integers). The position where the stirrer is provided is not limited to the above-mentioned position, and the installation position or number may be changed as necessary depending on the operation content of each step, or the stirrer may be provided for all inspection chips. It is also possible to provide it.

(Light projector)
The light projecting unit 20 includes a light source unit and a first angle adjusting unit (both not shown), and irradiates the inspection chip with excitation light.

The light source unit includes a light source, a beam shaping optical system, an APC mechanism, and a temperature adjusting mechanism, and irradiates the inspection chip with excitation light. 4A to 4C are schematic views showing the structure of the inspection chip 60. The details of the structure of the inspection tip 60 will be described later, but as shown in FIG. 4A, the inspection tip 60 includes a well body 61 and a side wall member 62, and is adjacent to the side wall member 62 as shown in FIGS. 4B and C. A second opening 64 is provided on the side wall of the well body 61. FIG. 5 is a partially enlarged cross-sectional view of the vicinity of the second opening 64 in the cross section of the inspection chip 60 in the height direction (vertical direction in FIG. 4), and is a schematic view showing the structure of the side wall member 62. The details of the structure of the side wall member 62 will be described later, but as shown in FIG. 5, the side wall member 62 includes a prism 71, a metal film 75, and a capture film 76, and the capture film 76 is formed in the second opening 64. It is exposed to form a reaction field 77.

FIG. 6 shows a cross section of the inspection chip 60, and is a schematic view showing light incident on the inspection chip 60 and light emitted from the inspection chip 60. As shown in FIG. 6, the light source unit emits excitation light 91 having a constant wavelength and light intensity so that the shape of the irradiation spot on the reflecting surface 73 of the prism 71 is substantially circular with respect to the prism 71 of the inspection chip 60. Irradiate. The size of the irradiation spot is preferably smaller than the reaction field 77.

The type of light source is not particularly limited, and is, for example, a laser diode (LD). Other examples of light sources include light emitting diodes, mercury lamps, and other laser light sources. When the light emitted from the light source is not a beam, the light emitted from the light source is converted into a beam by a lens, a mirror, a slit, or the like. When the light emitted from the light source is not monochromatic light, the light emitted from the light source is converted into monochromatic light by a diffraction grating or the like. Further, when the light emitted from the light source is not linearly polarized light, the light emitted from the light source is converted into linearly polarized light by a polarizer or the like.

The beam shaping optical system includes, for example, a collimator, a bandpass filter, a linear polarizing filter, a half-wave plate, a slit, and a zoom means. However, the beam shaping optical system may be configured to include only a part of these. The collimator collimates the excitation light emitted from the light source. The bandpass filter makes the excitation light emitted from the light source narrow-band light having only the central wavelength. This is because the excitation light emitted from the light source has a slight wavelength distribution width. The linearly polarized light filter turns the excitation light emitted from the light source into fully linearly polarized light. The half-wave plate adjusts the polarization direction of the excitation light so that the P wave component is incident on the reflecting surface 73. The slit and the zoom means adjust the beam diameter, contour shape, and the like of the excitation light so that the shape of the irradiation spot on the reflecting surface 73 becomes a circle of a predetermined size.

The APC mechanism controls the light source so that the output of the light source is constant. Specifically, the APC mechanism detects the amount of light branched from the excitation light by a photodiode or the like, and controls the input energy by a regression circuit to control the output of the light source to be constant.

The temperature adjusting mechanism is, for example, a heater or a Peltier element. Since the wavelength and energy of the light emitted from the light source may fluctuate depending on the temperature, the temperature adjustment mechanism constantly controls the wavelength and energy of the light emitted from the light source by keeping the temperature of the light source constant. To do.

The first angle adjusting unit adjusts the incident angle α of the excitation light 91 with respect to the reflection surface 73 by relatively rotating the optical axis of the excitation light 91 and the inspection chip 60.

For example, the first angle adjusting unit scans the incident angle α by rotating the light source unit around an axis along the height direction of the inspection chip 60 (the axis perpendicular to the paper surface in FIG. 6). As a result, even if the incident angle α fluctuates due to the scanning described above, the position of the irradiation spot of the excitation light 91 on the reflection surface 73 is maintained almost unchanged.

In this way, the first angle adjusting unit scans the incident angle α of the excitation light 91, so that the enhancement angle is specified by the detection unit 40 described later. The augmentation angle is a plasmon scattered light 94 having the same wavelength as the excitation light 91, which is emitted to the well body 61 side of the inspection chip 60 through the reflection surface 73 when the reflection surface 73 is irradiated with the excitation light 91. This is the angle of incidence when the amount of light is maximized. The enhancement angle is set as the incident angle α of the excitation light 91 at the time of optical blank measurement and fluorescence value measurement, which will be described later. The incident conditions of the excitation light 91 such as the augmentation angle are determined by the design elements of the inspection chip 60 (for example, the material and shape of the prism 71, the film thickness of the metal film 75, the wavelength of the excitation light 91, etc.) and the inside of the inspection chip 60. It is generally determined by the refractive index of the contained liquid, but may vary depending on the shape error of the prism 71, the composition of the liquid contained in the inspection chip 60 (for example, the type and amount of the fluorescent substance, etc.). Therefore, it is preferable to specify the optimum enhancement angle for each inspection.

(Detection unit)
The detection unit 40 includes a first lens, an optical filter, a second lens, a position switching means, and a light receiving sensor (all not shown), and includes fluorescence 93 and plasmon scattered light 94 emitted from the inspection chip 60. Is detected.

The first lens is, for example, a condensing lens, which condenses light emitted from the vicinity of the reaction field 77. The second lens is, for example, an imaging lens, and the light collected by the first lens is imaged on the light receiving surface of the light receiving sensor. The optical paths between the first lens and the second lens are substantially parallel optical paths.

When detecting fluorescence 93, the optical filter is arranged on the optical path between the first lens and the second lens by the position switching unit. The optical filter is, for example, a filter including a multilayer film that reflects a predetermined light component, or a colored glass filter that absorbs a predetermined light component, and among the light focused by the first lens, excitation light 91 or plasmon. The excitation light component such as the scattered light 94 is removed, and only the fluorescence 93 is guided to the light receiving sensor. As a result, the light receiving sensor can detect the fluorescence 93 with a high S (signal) / N (noise) ratio. Examples of optical filters include excitation light reflection filters, short wavelength cut filters and bandpass filters.

When detecting the plasmon scattered light 94, the optical filter is arranged outside the optical path between the first lens and the second lens. In this case, the augmentation angle, which is the incident angle when the amount of light of the plasmon scattered light 94 is maximized, is specified.

The position switching unit arranges the optical filter on the optical path between the first lens and the second lens or outside the optical path, if necessary. Specifically, when detecting the fluorescence 93, the optical filter is arranged on the same optical path, and when detecting the plasmon scattered light 94, the optical filter is arranged outside the same optical path.

The light receiving sensor detects fluorescence 93 and plasmon scattered light 94. The light receiving sensor is, for example, a photomultiplier tube (PMT) or an avalanche photodiode (APD). However, the light receiving sensor is not limited to these, and may be any one capable of detecting weak fluorescence 93 and having high sensitivity.

Further, the detection unit 40 may be configured to detect the reflected light 92 of the excitation light 91 instead of detecting the plasmon scattered light 94. For example, the light receiving sensor may be used, or a light receiving sensor (for example, a photodiode) for detecting reflected light may be separately provided to detect the reflected light 92. In this case, when the first angle adjusting unit of the light projecting unit 20 scans the incident angle α of the excitation light 91, the detection unit 40 specifies the resonance angle instead of the augmented angle, and the optical blank measurement and the optical blank measurement described later are performed. It is set as the incident angle α of the excitation light 91 when measuring the fluorescence value. The resonance angle is an angle of incidence when the amount of reflected light 92 of the excitation light 91 reflected by the reflection surface 73 is minimized when the reflection surface 73 is irradiated with the excitation light 91. The resonance angle exists in the very vicinity of the augmentation angle.

Further, the light projecting unit 20 and the light receiving sensor are arranged at the same height as the inspection chip 60. As a result, the biochemical inspection system can be miniaturized. However, the light projecting unit 20 and the light receiving sensor do not necessarily have to be arranged at the same height as the inspection chip 60. For example, it is possible to freely change the positions of the light emitting unit 20 and the light receiving sensor by using a mirror or the like.

(Liquid transfer / transport section)
The liquid transfer / transfer unit 30 includes a liquid transfer means and a transfer means (both not shown). The liquid feeding means supplies a liquid such as a reagent to the inspection chip as needed, and collects the liquid contained in the inspection chip. The transport means moves the inspection chip and places it in an appropriate position as needed.

The liquid feeding means includes a reagent tip, a pipette unit, and a first moving mechanism (all not shown).

The reagent chip is a container that can contain a sample, a sample dilution solution, a measurement buffer solution, a washing solution, a labeling solution for imparting a fluorescent label to a substance to be detected, and the like. The types of the sample and the substance to be detected are not particularly limited. Examples of specimens include body fluids such as blood, serum, plasma, cerebrospinal fluid, urine, nasal fluid, saliva, semen and tissue extracts. Examples of substances to be detected include nucleic acids (DNA and RNA), proteins (polypeptides, oligopeptides, etc.), amino acids, sugars, lipids and modified molecules thereof. The sample dilution solution comprises, for example, BSA (bovine serum albumin), Antifoam SI, NaN3, CMD (carboxymethyl-dextran), HAMA (human anti-mouse antibodies) inhibitor, and PBST (phosphate buffered saline with Tween 20). The measurement buffer solution comprises, for example, BSA, Antifoam SI, NaN3, PBST. It consists of a cleaning solution, for example Antifoam SI, NaN3, PBST. The labeling solution comprises, for example, a secondary antibody labeled with a fluorescent substance and PBST. In addition, the reagent chip is usually composed of a plurality of containers arranged according to the type of liquid, or a plurality of containers integrated.

The pipette unit consists of a syringe pump and a nozzle. The syringe pump includes a syringe, a plunger capable of reciprocating in the syringe, and a drive mechanism, and quantitatively sucks or discharges a liquid by the reciprocating motion of the plunger. The drive mechanism is a means for reciprocating the plunger, and includes, for example, a stepping motor. One end of the nozzle is connected to the syringe pump. A pipette tip is attached to the other end of the nozzle that is not connected to the syringe pump. However, without using a pipette tip, it is also possible to directly supply a liquid such as a reagent into the inspection tip by a nozzle, or to directly collect the liquid contained in the test tip by a nozzle.

The first moving mechanism moves the nozzle and arranges it at a predetermined position. For example, the first moving mechanism freely moves the nozzle in two directions, a vertical direction and a horizontal direction. Examples of the first moving mechanism include a robot arm and a two-axis stage or a vertically movable turntable.

The transport means includes an inspection chip holding portion and a second moving mechanism (both not shown).

The inspection chip holding portion is for holding the inspection chip 60, and is fixed to or detachably attached to the second moving mechanism. The second moving mechanism moves the inspection chip holding portion to move the inspection chip 60 held by the inspection chip holding portion to positions 10a to 10e corresponding to each measuring unit that performs each step constituting the inspection. , Place it in the appropriate position if necessary. Examples of the second moving mechanism include a conveyor and a rotating stage. However, when a continuous form in which a plurality of inspections can be performed at substantially the same time is not adopted and a discontinuous form in which individual steps constituting one inspection are performed at the same position is adopted, it is not necessary to provide a second moving mechanism. For example, the second moving mechanism may be omitted, and only the inspection chip holding portion may be provided to hold the inspection chip 60. Further, even when the continuous form is adopted, if it is not necessary to move the inspection chip 60 according to the progress of the inspection, a plurality of inspection chips corresponding to each measurement unit that performs each step constituting one inspection. 60 is provided, and when the work in one measurement unit is completed, the liquid such as the reagent in the inspection chip 60 is moved into the inspection chip 60 corresponding to the next measurement unit by a liquid feeding means or the like instead of moving the inspection chip 60. It is possible to change. In this case as well, the second moving mechanism may be omitted, and only the inspection chip holding portion may be provided to hold each inspection chip 60.

(Control calculation unit)
FIG. 7 is a block diagram of the control calculation unit 50. As shown in FIG. 7, the control calculation unit 50 includes a CPU 51, a light projection control unit 52, a liquid feed drive control unit 53, a liquid feed movement control unit 54, a transfer control unit 55, and a detection control unit 56. , Computation unit 57.

The CPU 51 controls the entire measurement, and operates each control unit or calculation unit described later as necessary. The projection control unit 52 controls the projection unit 20 and irradiates the excitation light at a predetermined position. The liquid feed drive control unit 53 controls the pipette unit of the liquid feed means of the liquid feed / transport unit 30, and sucks or discharges a predetermined liquid in a predetermined amount. The liquid feed movement control unit 54 controls the first movement mechanism of the liquid feed means of the liquid feed / transport unit 30, and arranges the nozzle at a predetermined position. The transfer control unit 55 controls the transfer means of the liquid transfer / transfer unit 30, and arranges the inspection chip at an appropriate position as needed. The detection control unit 56 controls the detection unit 40 and detects plasmon scattered light or fluorescence as needed. The calculation unit 57 specifies the enhancement angle based on the amount of plasmon scattered light, performs quantitative measurement such as calculation of the concentration of the substance to be detected based on the amount of fluorescence light, and performs other data correction processing and the like.

(Inspection chip)
4A to 4C are schematic views showing the structure of the inspection chip 60. FIG. 4A is a perspective view of the inspection chip 60. As shown in FIG. 4A, the inspection tip 60 includes a well body 61 and a side wall member 62. The well body 61 has a bottomed structure capable of accommodating a liquid.

(Well body)
FIG. 4B is a perspective view of the well body 61, and FIG. 4C is a perspective perspective view of the well body 61. As shown in FIGS. 4B and 4C, the well body 61 has a first opening 63 at one end, a second opening 64 on the side wall adjacent to the side wall member 62, and is opposite to the first opening 63 side. It has a bottom structure 66 at the bottom edge on the side. Further, the well body 61 is a substantially cylindrical body in which the outer wall on the side on which the side wall member 62 is arranged is cut into a flat surface according to the width of the side wall member 62, and the bottom end is closed by the bottom structure 66. The space inside the well body 61 connected to the first opening 63 and the second opening 64 serves as a liquid storage portion 65 for storing a liquid such as a reagent. However, the shape of the well body 61 is not limited to a cylinder, and may be, for example, a square cylinder having a square cross section or a shape having an asymmetric cross section. In particular, when the well body 61 has a vertically long shape, the effect of further improving the detection accuracy of the substance to be detected and simultaneously achieving stable and efficient stirring is remarkable. Further, the shape of the outer wall of the well body 61 on the side where the side wall member 62 is arranged is not limited to a flat surface as long as the side wall member 62 can be fixed.

The well body 61 is made of a material that is transparent to light having a wavelength of excitation light 91 and light having a wavelength of fluorescence 93, and is made of, for example, resin or glass. However, a part of the well body 61 may be formed of a material opaque to the light having the wavelength of the excitation light 91 and the light having the wavelength of the fluorescence 93 as long as the measurement in the inspection method described later is not hindered.

The bottom surface structure 66 has a curved surface in which the tip end 66a is biased toward the side wall member 62. FIG. 6 is a schematic view of the inspection chip 60 when viewed from the first opening 63 side. As shown in FIG. 6, the tip 66a is not located at the center of symmetry c in the cross section of the well body 61, but at the tip position x biased toward the side wall member 62 from the center of symmetry c. The tip position x and the center of gravity of the inspection chip 60 in a state of containing a liquid such as a reagent (hereinafter, simply referred to as “the center of gravity of the inspection chip 60”) G2 are on the same axis in the length direction of the inspection chip 60. Located in. FIG. 8 is a schematic view for explaining the circular motion of the inspection tip 60 installed on the rotating body 99 of the stirring device. The upper part of FIG. 8 shows a side view of the inspection chip 60 installed on the rotating body 99 of the stirring device when stirring the liquid in the inspection chip 60. The lower part of FIG. 8 shows a schematic view when the inspection chip 60 is viewed from the first opening 63 side when the liquid in the inspection chip 60 is agitated. As shown in FIG. 8, since the inspection chip 60 and the rotating body 99 are in contact with each other at the tip 66a, the tip 66a is on the axis in the length direction of the inspection chip 60 where the center of gravity G2 of the inspection chip 60 exists. As shown in the lower part of FIG. 8, the center of gravity G2 of the inspection chip 60 and the tip 66a perform a circular motion in the same motion locus, and the inspection chip 60 makes a circular motion of the rotating body 99. It becomes possible to perform a stable circular motion with it. In the lower part of FIG. 8, for convenience of notation, the inspection chip 60 is indicated by a straight line, and the center of gravity of the inspection chip 60 and the motion locus of the tip 66a are indicated by broken lines, respectively.

As described above, if the inspection chip 60 can perform a stable circular motion, the inspection chip 60 will not fall from the stirring device, and the reagents and the like contained in the inspection chip 60 will be affected by the circular motion. The liquid can be efficiently agitated, and the liquid such as a reagent can be sufficiently supplied to the reaction field described later.

However, the bottom surface structure 66 is not limited to a curved surface, and may be, for example, a pyramid having a tip at the tip position x or a flat surface having a protrusion at the tip position x. That is, the bottom surface structure 66 may be configured to come into contact with the rotating body 99 of the stirring device at the tip position x and receive a circular motion. Further, from the viewpoint of stability when mounted on the rotating body 99, the bottom surface structure 66 preferably has the same shape as the surface of the rotating body 99 in contact with the bottom structure 66.

Further, in the present embodiment, the tip position x and the center of gravity G2 of the inspection chip 60 are located on the same axis in the length direction of the inspection chip 60, but the tip position x is not limited to this. The well body 61 may be biased toward the side wall member 62 from the center position of the cross section (center of symmetry c in this embodiment). For example, due to the weight of the side wall member 62 or the like, it is impossible due to the structure of the inspection chip 60 to arrange the tip position x and the center of gravity G2 of the inspection chip 60 on the same axis in the length direction of the inspection chip 60. In some cases. In that case, the tip position x and the center of gravity G2 of the inspection chip 60 do not necessarily have to be located on the same axis in the length direction of the inspection chip 60, and the tip position x is arranged so as to be biased toward the side wall member 62 side. Then, the effect of stabilizing the circular motion of the inspection chip 60 and efficiently stirring the liquid such as the reagent contained in the inspection chip 60 can be obtained.

Further, as described above, the center of gravity of the inspection chip 60 is exactly the center of gravity of the inspection chip 60 in a state where a liquid such as a reagent is contained, but for convenience of manufacturing or the like, the center of gravity of the inspection chip 60 is used as the inspection chip. It can also be the center of gravity of the 60 itself.

(Wall member)
FIG. 5 is a partially enlarged cross-sectional view of the vicinity of the second opening 64 in the cross section of the inspection chip 60 in the height direction (vertical direction in FIG. 4), and is a schematic view showing the structure of the side wall member 62. As shown in FIG. 5, the side wall member 62 includes a prism 71, a metal film 75, and a capture film 76, and the capture film 76 is exposed at the second opening 64 to form a reaction field 77. The side wall member 62 is adhered to the well body 61 via an adhesive layer (not shown) so that the second opening 64 can be closed without leaking a liquid such as a reagent contained in the inspection chip 60. However, the side wall member 62 may be joined to the well body 61 by laser welding, ultrasonic welding, crimping using a clamp member, or the like without using an adhesive layer.

The prism 71 is an optical element made of a dielectric material transparent to the excitation light 91, and has not a little birefringence characteristic. Examples of the material of the prism 71 include a resin and glass, preferably a resin having a refractive index of 1.4 to 1.6 and a small birefringence.

FIG. 6 is a schematic view of the inspection chip 60 when viewed from the first opening 63 side, and is a schematic view showing the light incident on the inspection chip 60 and the light emitted from the inspection chip 60. As shown in FIG. 6, the prism 71 is a pillar body having a trapezoid as a bottom surface, the surface corresponding to one base of the trapezoid is the reflecting surface 73, the surface corresponding to one leg is the incident surface 72, and the other. The surface corresponding to the leg is the exit surface 74. The excitation light 91 emitted from the light projecting unit 20 is incident on the incident surface 72. In the prism 71, the light that has passed through the incident surface 72 and has entered the inside of the prism 71 is reflected by the reflecting surface 73, and the reflected light 92 reflected by the reflecting surface 73 passes through the emitting surface 74 to the outside of the prism 71. It is configured to emit light. However, the shape of the prism 71 is not limited to a column having a trapezoid as a bottom surface, and may be, for example, a triangular prism or a semi-cylinder. Further, the reflecting surface 73 is preferably flat.

Further, when the light source of the excitation light 91 is a laser diode (LD), when the excitation light 91 returns to the LD, the excited state of the LD is disturbed and the wavelength and output of the excitation light 91 fluctuate. The surface 72 is formed so that the excitation light 91 does not return to the light projecting unit 20, and the angle with the reflecting surface 73 is set so that the excitation light 91 does not enter the incident surface 72 perpendicularly. In the present embodiment, the angle between the incident surface 72 and the reflecting surface 73 and the angle between the reflecting surface 73 and the emitting surface 74 are both about 80 °.

The metal film 75 is formed on the reflecting surface 73 of the prism 71. The material of the metal film 75 is not particularly limited as long as it is a metal capable of causing surface plasmon resonance. Examples of materials for the metal film 75 include gold, silver, copper, aluminum and alloys thereof. The method for forming the metal film 75 is not particularly limited. Examples of methods for forming the metal film 75 include sputtering, vapor deposition, and plating. The thickness of the metal film 75 is not particularly limited, but is preferably in the range of 30 to 70 nm.

The capture film 76 is a region on the metal film 75 on which the first capture body that specifically binds to the substance to be detected is immobilized. The type of the first trap is not particularly limited as long as it can specifically bind to the substance to be detected. Examples of the first trap include an antibody (primary antibody) that can specifically bind to the substance to be detected or a fragment thereof, a nucleic acid, an enzyme, or the like.

The reaction field 77 is a region of the capture membrane 76 exposed in the liquid storage portion 65 of the well body 61 at the second opening 64. In the reaction field 77, the first trapped substance immobilized on the metal film 75 to form the trapping film 76 selectively binds the substance to be detected present in the sample to selectively bind the substance to be detected. Capture. From the viewpoint of detection accuracy, the surface on which the reaction field 77 is formed, that is, in the present embodiment, the surface of the region of the metal film 75 corresponding to the reaction field 77 is preferably a flat surface. Further, a protective layer for maintaining the capture ability of the first capture body for a long period of time may be applied on the reaction field 77.

The size of the reaction field 77 is not particularly limited. When the capture membrane 76 is large enough to block the second opening 64, the size of the reaction field 77 is defined by the second opening 64. Thereby, the size of the reaction field 77 can be easily adjusted with high accuracy. On the other hand, when the capture film 76 is smaller than the second opening 64, the size of the capture film 76 becomes the size of the reaction field 77 as it is.

Further, the reaction field 77 is preferably arranged at a position away from the bottom surface of the well body 61 on the bottom surface structure 66 side. As a result, a liquid such as a reagent can be supplied to the reaction field 77 in the liquid storage unit 65 to efficiently carry out the reaction. Further, when detecting the fluorescence 93, it is possible to prevent the detection accuracy from being lowered due to noise caused by the bottom surface of the well body 61 on the bottom surface structure 66 side.

(Operation of biochemical inspection system)
FIG. 9 is a flowchart for explaining the operation of the biochemical inspection system A. The operation of the biochemical inspection system A will be described with reference to FIG.

First, preparation for measurement is performed (step S10). Specifically, under the control of the control calculation unit 50, the liquid feeding / transporting unit 30 moves the target inspection chip 60 to the position 10a (see FIG. 3) of the biochemical inspection system A, and moves the inspection chip 60 to the position 10a. Attach to the rotating body of the corresponding agitator. Then, the cleaning liquid is supplied to the inspection chip 60 by the liquid feeding / transporting unit 30, and the inside of the liquid accommodating unit 65 is cleaned while the liquid in the inspection chip 60 is agitated by the vibrating unit 10. At this time, if a protective layer for maintaining the capturing ability of the first trapping material for a long period of time is applied on the reaction field 77, the protective layer is also removed. After that, the cleaning liquid in the inspection chip 60 is collected by the liquid feeding / transporting unit 30, and the measurement buffer solution is newly supplied into the inspection chip 60.

Next, the inspection chip 60 is irradiated with excitation light to perform augmentation measurement for specifying the augmentation angle and optical blank value measurement for measuring the optical blank value (step S20). Specifically, under the control of the control calculation unit 50, the liquid feeding / transporting unit 30 arranges the target inspection chip 60 at the position 10b (see FIG. 3) of the biochemical inspection system A, and the light projecting unit 10 inspects. The region of the reflecting surface 73 corresponding to the reaction field 77 of the chip 60 is irradiated with the excitation light 91 while scanning the incident angle α. At the same time, the detection unit 40 detects the plasmon scattered light 94 emitted from the metal film 75 irradiated with the excitation light 91 to the inside of the inspection chip 60. The control calculation unit 50 acquires data including the relationship between the incident angle α of the excitation light 91 and the intensity of the plasmon scattered light 94, and based on the data, the incident when the intensity of the plasmon scattered light 94 is maximized. The angle α is specified as the augmentation angle, and the incident angle α of the excitation light 91 is set as the augmentation angle. The enhancement angle is determined on the order of about 0.1 °.

After that, under the control of the control calculation unit 50, the light projecting unit 10 irradiates the region of the reflecting surface 73 corresponding to the reaction field 77 of the inspection chip 60 with the excitation light 91 at the incident angle α set to the augmentation angle. At the same time, the detection unit 40 detects the amount of light having the same wavelength as the fluorescence 93. The control calculation unit 50 records the amount of light measured by the detection unit 40 as an optical blank value.

After that, the liquid feeding / transporting unit 30 collects the measurement buffer solution in the test chip 60, and the measurement sample is newly supplied into the test chip 60. As the sample for measurement, a sample directly collected from the examinee may be used, or a sample directly collected from the examinee diluted with a sample dilution solution may be used.

Next, a primary reaction for binding the substance to be detected present in the sample to the first trap exposed in the reaction field 77 is performed (step S30). Specifically, under the control of the control calculation unit 50, the liquid feeding / transporting unit 30 moves the target inspection chip 60 to the position 10c (see FIG. 3) of the biochemical inspection system A, and moves the inspection chip 60 to the position 10c. Attach to the rotating body of the corresponding agitator. Then, the liquid in the inspection chip 60 is agitated by the vibrating unit 10. At this time, the substance to be detected present in the sample specifically binds to the first trap exposed in the reaction field 77, so that the substance to be detected is captured by the first trap and stays in the reaction field 77. ..

After a sufficient time has elapsed for the reaction, in order to clean the inside of the inspection chip 60, the measuring sample in the inspection chip 60 is collected by the liquid feeding / transporting unit 30, and the cleaning liquid is newly supplied into the inspection chip 60. Will be done. At this time, since the liquid in the inspection chip 60 is continuously agitated by the vibrating unit 10, the substances to be detected, impurities, and the like that are non-specifically adsorbed in the inspection chip 60 are removed.

After that, the cleaning liquid in the inspection chip 60 is collected by the liquid feeding / transporting unit 30, and the labeling liquid is newly supplied into the inspection chip 60.

Next, a secondary reaction for imparting a fluorescent label to the substance to be detected captured by the first trap is carried out (step S40). Specifically, under the control of the control calculation unit 50, the liquid feeding / transporting unit 30 moves the target inspection chip 60 to the position 10d (see FIG. 3) of the biochemical inspection system A, and moves the inspection chip 60 to the position 10d. Attach to the rotating body of the corresponding agitator. Then, the liquid in the inspection chip 60 is agitated by the vibrating unit 10. In the labeling solution, there is a fluorescently labeled second trap, and the second trap is a substance to be detected at a site different from the site of the substance to be detected that specifically binds to the first trap. By specifically binding to the second trap, the substance to be detected is indirectly fluorescently labeled. The type of the second trap is not particularly limited as long as it can specifically bind to the substance to be detected at a site different from the site of the substance to be detected that specifically binds to the first trap. .. For example, the second trap may be a biomolecule specific to the substance to be detected, a fragment thereof, or the like. Further, the second trap may be one composed of one molecule or a complex formed by binding two or more molecules.

After a sufficient time has elapsed for the reaction, in order to clean the inside of the inspection chip 60, the labeling liquid in the inspection chip 60 is collected by the liquid feeding / transporting unit 30, and the cleaning liquid is newly supplied into the inspection chip 60. Ru. At this time, since the liquid in the inspection chip 60 is continuously agitated by the vibrating unit 10, the second trap, impurities, and the like that are non-specifically adsorbed in the inspection chip 60 are removed.

After that, the cleaning liquid in the inspection chip 60 is collected by the liquid feeding / transporting unit 30, and the measurement buffer solution is newly supplied into the inspection chip 60.

Next, the fluorescence value measurement for measuring the fluorescence value from the fluorescently labeled substance to be detected is performed (step S50). Specifically, under the control of the control calculation unit 50, the liquid feeding / transporting unit 30 arranges the target inspection chip 60 at the position 10e (see FIG. 3) of the biochemical inspection system A, and the light projecting unit 10 inspects. The region of the reflection surface 73 corresponding to the reaction field 77 of the chip 60 is irradiated with the excitation light 91 at the incident angle α set at the augmentation angle. At the same time, the detection unit 40 detects the amount of light having the same wavelength as the fluorescence 93. The control calculation unit 50 records the amount of light measured by the detection unit 40 as a fluorescence value. At this time, if the liquid level of the liquid (buffer solution for measurement) in the liquid storage unit 65 and the position of the reaction field 77 are close to each other, even the fluorescence reflected or refracted on the liquid surface is detected by the detection unit 40. From the viewpoint of detection accuracy, the reaction field 77 is preferably located below the liquid level of the liquid (buffer solution for measurement) in the liquid storage portion 65 and at a position away from the liquid level. Therefore, the measurement buffer solution in this step may be supplied in a larger amount than the liquid used in other steps.

After that, the inspection chip 60 is disposed of under the control of the control calculation unit 50, and the control calculation unit 50 correlates with the amount of the substance to be detected by subtracting the optical blank value acquired in step S20 from the acquired fluorescence value. Calculate the signal value to be used. Further, the control calculation unit 50 may further convert the signal value into the amount or concentration of the substance to be detected based on the calibration curve prepared in advance.

After that, the inspection is completed. Through each of the above steps, the biochemical test system A can measure the presence or amount of the substance to be detected in the sample.

In the step S20, the incident angle α of the excitation light 91 is set as the augmentation angle, but instead of the augmentation angle, the incident angle α of the excitation light 91 may be set as the resonance angle. In this case, in step S20, the light projecting unit 10 irradiates the region of the reflecting surface 73 corresponding to the reaction field 77 of the inspection chip 60 with the excitation light 91 while scanning the incident angle α. At the same time, the detection unit 40 detects the amount of reflected light 92. The control calculation unit 50 acquires data including the relationship between the incident angle α of the excitation light 91 and the light amount of the reflected light 92, and based on the data, the incident angle α when the light amount of the reflected light 92 is minimized. Is specified as the resonance angle, and the incident angle α of the excitation light 91 is set as the resonance angle.

This application claims priority under Japanese Patent Application No. 2017-025823 filed on February 15, 2017. All the contents described in the application specification and drawings are incorporated herein by reference.

10 Vibration unit 10a, 10b, 10c, 10d, 10e Position 20 Light projector 30 Liquid transfer / transfer unit 40 Detection unit 50 Control calculation unit 51 CPU
52 Light projection control unit 53 Liquid feed drive control unit 54 Liquid feed movement control unit 55 Transport control unit 56 Detection control unit 57 Calculation unit 60, 60a, 60b, 60c, 60d, 60e, 60x, 60y Inspection chip 61 Well body 62 Side wall Member 63 1st opening 64 2nd opening 65 Liquid storage 66 Bottom structure 66a, 66b Tip 71 Prism 72 Incident surface 73 Reflection surface 74 Exit surface 75 Metal film 76 Capture film 77 Reaction field 91 Excitation light 92 Reflection light 93 Fluorescence 94 Plasmon Scattered light 99 Rotating body A Biochemical inspection system c Center of symmetry x Tip position G1, G2 Center of gravity α Incident angle

Claims (12)

An inspection chip that stores a liquid inside and agitates the liquid by circular motion at the bottom edge.
It has a well body for accommodating the liquid and a side wall member arranged on the side surface of the well body.
The bottom end of the well body has a bottom structure in contact with a rotating member for circularly moving the bottom end at a position biased from the center line of the well body to the side wall member.
Inspection tip. The bottom surface structure is formed so that the position of contact with the rotating member is located on an axis parallel to the center line and the center of gravity of the entire inspection chip is present.
The inspection chip according to claim 1. The inspection chip according to claim 1 or 2, wherein the shape of the bottom surface structure is a curved surface. The inspection chip according to any one of claims 1 to 3, wherein the well body has a vertically long shape. The inspection chip according to claim 4, wherein the well body has a tubular shape whose bottom end is closed by the bottom structure. The inspection chip according to claim 5, wherein the well body is a cylinder. The inspection chip according to any one of claims 1 to 6, wherein the side wall member includes an optical element. The inspection chip according to claim 7, wherein the side wall member includes a prism. The well body has a through hole on the side surface and has a through hole.
A metal film is formed on one surface of the prism.
The metal film is exposed inside the well body in the through hole.
The inspection chip according to claim 8. The test chip according to claim 9, wherein a ligand molecule for capturing a substance to be detected is immobilized on the metal film exposed inside the well body. An inspection system using the inspection chip according to any one of claims 1 to 10.
A light source that irradiates the inspection chip with light,
A detection unit for measuring the light to be measured emitted from the inspection chip,
A stirring device having the rotating member and
Inspection system with. Further having a transport unit for transporting the inspection chip,
The transport unit transports the inspection chip and arranges it at a predetermined position according to the progress of the inspection.
The inspection system according to claim 11.

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